Project summary. The islet of Langerhans is a multi-cellular micro-organ located in the pancreas. It is central to maintaining blood glucose homeostasis through secretion of hormones such as insulin and glucagon. Interactions between cells in the islet are crucial to the regulation of glucose stimulated insulin secretion (GSIS). Beta cells within the islet secrete insulin with a many-fold increase in GSIS response compared to isolated beta cells. Furthermore beta cells in the islet exhibit synchronized oscillations which lead to pulsatile insulin from the whole pancreas. These coordinated insulin pulses are thought to be more effective in lowering blood glucose and maintaining insulin sensitivity. The importance of the structure and signaling within the islet is highlighted by the fact that type 1 diabetes can be effectively reversed by the transplantation of intact islets (but not by isolated beta cells). Previous research and preliminary data shows that gap junction coupling has a physiological role in islet function. However other research has also shown possible roles for paracrine and juxtacrine mechanisms in cell-cell communication. We hypothesize that it is primarily gap junction coupling of electrical activity which serves to coordinate and enhance GSIS, but coupling of cAMP mediated signaling between alpha and beta cells within the islet can further modulate GSIS. To test this hypothesis it will be necessary to introduce precise experimental perturbations, and use quantitative techniques to monitor the resulting impact on signaling pathways underlying GSIS. During the mentored phase of this proposal we will establish the necessary physiological and biochemical assays, as well as to refine the quantitative mathematical models currently in use. Two specific aims are then proposed for the independent research phase: 1) quantify the relative role of gap junction coupling and KATP channel regulated membrane polarization in regulating GSIS, 2) determine the mechanism and role for coupling of cAMP signaling between beta cells and alpha cells within the islet. These two aims can proceed independently, although the results from each aim will be complimentary to testing the overall hypothesis. Experiments will be performed on a number of gap junction knockout and KATP channel transgenic mouse models as well as human islets, and will utilize state-of- the art quantitative imaging approaches, fluorescent protein biosensors and mathematical models along with more established biochemical and physiological assays. These experiments will yield a more complete understanding of signaling mechanisms within the islet which will be important for the development of transplantation and stem cell therapies for type1 diabetes as well as identifying novel therapeutic drug targets for type2 diabetes. PUBLIC HEALTH RELEVANCE: Relevance. The pancreatic islet plays a central role in glucose homeostasis and the failure of the islet leads to the development of diabetes. Communication between cells is crucial to the function of the islet, but knowledge of the mechanisms involved is unclear. A precise understanding of the signaling between different cells within the islet will improve the development of transplantation and stem cell therapies and identify drug targets to effectively treat individuals with diabetes.